Porin

Individual dissected posterior eyecups were lysed in 160 μL of HNTG detergent buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl₂, 1% Triton X-100) freshly supplemented with 1% protease and phosphatase inhibitor cocktails (#P8340, Sigma-Millipore, and #78420, Thermofisher, respectively). Cleared lysates representing equal eyecup tissue fractions were separated on 12% SDS polyacrylamide gels using a standard Tris–glycine buffer system (Novex gels and solutions, Thermofisher). Proteins were transferred to nitrocellulose membranes (#88018, Thermofisher) for immunodetection with primary and appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. The primary antibodies used were porin (#4866, Cell Signaling, 1:5,000), RhoA (#sc-179, Santa Cruz Biotechnologies, Santa Cruz, CA, USA, 1:200), rhodopsin (clone B6-30, 0.01 μg/mL), and α-tubulin (#9099, Cell Signaling, 1:5,000). The secondary antibodies used were donkey-anti-rabbit IgG-HRP (#16029, Thermofisher, 1:10,000) and donkey-anti-mouse IgG-HRP (#16011, Thermofisher, 1:5,000). Chemiluminescence reaction signals from ECL-plus substrate (#NEL105001EA, Perkin-Elmer, Shelton, CT, USA) were captured with X-ray films (#3018, Denville Scientific, Swedesboro, NJ, USA), which were scanned and quantified by densitometry using Image Quant^TM TL 7.0 (GE Healthcare, Chicago, IL, USA).